Several methods are currently in use for delivering defined foreign DNA segments into eukaryotic cells. These include physical injection of DNA (Mueller et al., Cell, 15:579-585, 1978); fusion of DNA-containing liposomes (Ostro et al., Nature, 274:921-923, 1978; Dimitriadis, Nature, 274:923-924) or erythrocytes (Rechsteiner, Natl. Cancer Inst. Monogr., 48:57-64, 1978) with target cells; and the direct application of naked DNA onto cells in the presence of calcium phosphate (Graham et al., Virology, 52:456-461, 1973) and marker DNA (Wigler et al., Cell, 11:223-232, 1977; Manel et al., Nature, 281:40-46, 1979; Wold et al., Proc. Natl. Acad. Sci. USA, 76:5684-5688, 1979).
More recently, insertion of DNA into recipient cells was achieved by using viral particles (SV40) in which a segment of the viral genome is covalently linked to defined nucleic acid segments (Mulligan et al., Nature, 277:108-114, 1979; Hammer et al., Nature, 281:35-40, 1979; Gruss and Khoury, Proc. Natl. Acad. Sci. USA, 1980 in press). While the SV40 vector system offers a rapid and efficient way to introduce foreign DNA's into permissive host cells, the system is limited by the size of DNA that can be accommodated within the virus particle. Moreover, since monkey cells are permissive for SV40 replication, infection by recombinant-SV40 particles culminates in cell death. SV40 DNA has not yet been exploited as a cloning vector in non-permissive rodent cells because (a) SV40 transformation is associated with integration of the viral genome, a process that may disrupt the integrity of the foreign DNA segment of interest, and (b) there is no indication that the gene will be active at detectable levels from the low integrated copy numbers which are sufficient for the expression of the SV40 transforming gene.
The present inventors have obtained good results utilizing certain papilloma virus genomes. Previously, studies on the molecular biology and genetics of the various papilloma viruses have been limited due to the lack of a cell-culture system suitable for propagating these viruses. However, studies on those papilloma viruses capable of inducing malignant transformation of eukaryotic cells in tissue culture indicate that the papillomavirus DNA is stably associated with the cell in an extrachromosomal state. Furthermore, evidence exists that the DNA of papilloma viruses not capable of inducing malignant transformation in eukaryotic cells remain stably associated with the cells (Lancaster and Meinke, Nature, 256:434-436, 1975).
Papers related to the biology of papilloma viruses and pipilloma virus DNA are listed below.
Lancaster, W. P., and W. Meinke, Nature, 256:434-436, 1975.
Lowry, D. R., et al, Nature, 287:72-74, 1980.
Howley, P. M., et al, Viruses in Naturally Occurring Cancers, Cold Spring Harbor Laboratory, pages 233-247, 1980.
Heilman, C. A., et al, J. Virology, 36:395-407, 1980.
The present invention provides unique vectors, segments of papilloma virus DNA for introducing foreign DNA into eukaryotic cells without the problems encountered in the prior art.
In the system described herein viral replication does not result in the death of the cell; many copies of the recombinant DNA are present per cell; by virtue of the transformed phenotype imparted by the bovine papilloma virus (BPV) DNA component no additional identification steps are required for identifying cells which have incorporated the foreign gene; the recombinant DNA exists exclusively as unintegrated extrachromosomal molecules; hence, the physical integration of the foreign gene is preserved and cells containing the foreign gene can be propagated indefinitely.